Project Summary/Abstract Integrins play important roles in basic cellular functions such as adhesion, growth, and migration as well as specialized functions in platelet plug formation during hemostasis and in T cell mediated immune responses. Abnormal expression and hyperactivity of integrins leads to cardiovascular diseases, impaired inflammatory responses, T cell malfunction, and enhanced tumor metastasis. Current integrin inhibitors that block extracellular ligand binding often cause serious adverse effects. Our overall goal is to elucidate the molecular mechanisms that activate integrins more specifically through the inside-out signaling pathway, thus facilitating the development of new therapies targeting this pathway. The main focus of this proposed study is to understand the structural basis of intermolecular interactions and intramolecular rearrangements that modulate integrin activity, thus altering cell adhesion and motility. The inside-out integrin signaling pathway has recently emerged as a new target for suppressing integrin activity. Integrin activation through this pathway is triggered by talin and mediated by a small GTPase, Rap1, and its effector protein RIAM (Rap1-interacting adaptor molecule). A co-activator, kindlin, also significantly enhanced the integrin activity. However, many key questions regarding how specific inter- and intra-molecular interactions regulate inside-out integrin activation remain unanswered. We have previously determined the structural basis of RIAM recruitment by Rap1 and the PM translocation and conformational activation of talin by RIAM. In this proposal, we aim to address the following central questions regarding the conformational rearrangement and specific interactions underlying the functional regulation of RIAM, talin, and kindlin: 1) to elucidate the structural basis of regulatory intramolecular interactions of RIAM and talin; 2) to determine the molecular basis of enhanced talin activity induced by the interaction of RIAM and talin; and 3) to probe the molecular mechanisms underlying the different integrin- regulating properties among kindlin isoforms and the co-localization of kindlin and talin in integrin signaling. Our studies will significantly advance the understanding of the regulatory mechanisms of integrin activation through the inside-out pathway.